The present invention relates to Chlorella extracts for use as immunomodulators.
Chlorella is a unicellular green algae that has been called a sun-powered supernutrient. Attested beneficial properties of this edible microalgae include wound healing, detoxification, constipation relief and growth stimulation. A number of studies have also indicated that Chlorella has beneficial effects on the immune system, both in vitro and in vivo.
Chlorella occurs in both fresh water and marine water. Species of the Chlorella genus exhibit striking diversity of physiological and biochemical properties (Kessler, E. xe2x80x9cPhycotalkxe2x80x9d 1989, 1:141-153; V. Rastogi Publ., New Delhi, India). Chlorella produces little cellulose and other indigestible cell wall material, and hence has been extensively investigated as a possible new source of food, especially as feedstock (Lee, Robert E. xe2x80x9cPhycologyxe2x80x9d 2nd edition; 1989, page 281; Cambridge University Press).
Chlorella has the highest content of chlorophyll of any known plant. It also contains vitamins, minerals, dietary fibre, nucleic acids, amino acids, enzymes, etc. It contains more than 9% fats (out of which polyunsaturated fatty acids [PUFA] represent about 82%). The vitamin content consists of provitamin A, vitamins B1, B2, B6, niacin, B12, biotin, vitamin C, vitamin K, pantothenic acid, folic acid, choline, lipoic acid, ionositol, PABA etc. Among the minerals present the most important are P, K, Mg, S, Fe, Ca, Mn, Cu, Zn and Co. The main components of Chlorella cells are about 60% protein (composed of all basic amino acids), and 20% carbohydrate.
Aqueous extracts of Chlorella pyreinodosa has been used for its nutritive content as well as for other health benefits. Studies report on numerous health benefits including improved immune system function and detoxification of harmful toxins. It was introduced as a health food in the USA in 1977 when novel technology processes were developed which made it more digestible and has been the largest selling health food supplement in Japan for a number of years. The Taiwan Chlorella company is the world""s largest supplier of Chlorella, and sells the product worldwide to Asia, Europe and North America, under the following brand names: Algea, Bio-REURELLA, Green Gem, Green Boost, Green Nature, Green Power, Joyau Vert and Natural Boost.
A number of studies have documented C. vulgaris extracts which have anti-tumor activity, as well as activity against Listeria and E. coli (Tanaka et al. Immunopharmacol. Immunotoxicol., 1990, 12(2):277-291; Tanaka et al. Cancer Immunol. Immunother., 1998, 45(6):313-320; Hasegawa et al. Int. J. Immunopharmacol., 1990, 12(8):883-891). These activities appear to be immune-mediated rather than direct toxicity against the tumor or pathogen.
A number of Chlorella extracts are available commercially, including products by Swiss Herbal and Nature""s Way. The Swiss Herbal product is identified as pure Chlorella broken cells containing Protein 61%, Carbohydrate 21.1%, Fat 11.0%, Chlorophyll 2.866%, RNA 2.94% and DNA 0.28%.
Other publications related to the present field include the following:
Japanese patent application laid open No. Sho 58-15920 discloses polysaccharides from fresh water Chlorella having immune potentiator and anti-tumor activity.
Neveu et al. Experientia, 1978, 34(12):1644-1645 discloses that C. pyrenoidosa is an immune response modulator.
Vermeil and Morin CR Seances Soc. Biol. Fil., 1976, 170(3):646-649 discloses that C. pyrenoidosa, presumably by nature of its cell wall, protects mice against sarcoma grafting.
Miyazawa et al. J. Ethnopharmacol., 1988, 24(2-3):135-146 discloses that C. pyrenoidosa cells or extract mediate host immune enhancement of the anti-tumor response.
Umezawa et al. Chemotherapy, 1982, 30(9):1041-1046 and Komiyama et al. Chemotherapy, 1986, 34:302-307 disclose that the acidic polysaccharide Chlon A from C. pyrenoidosa has immune enhancing and anti-tumor effects. Chlon A contains rhamnose, arabinose, glucose, galactose and glucuronic acid.
White and Barber Biochimica Biophysica Acta, 1972, 264:117-128 discloses an 88 kDa acidic polysaccharide from C. pyrenoidosa containing mainly rhamnose, as well as arabinose, galactose, xylose, mannose and glucuronic acid.
U.S. Pat. No. 4,533,548 discloses acidic polysaccharide CH-1 from C. pyrenoidosa containing mainly rhamnose, as well as arabinose, galactose, glucose and glucuronic acid. The polysaccharide was obtained via gel filtration with Sephadex G-75.
U.S. Pat. No. 4,831,020 discloses a polysaccharide extract from C. minutissima, a marine Chlorella, with immune-stimulating and anti-tumor activity. This patent states that polysaccharides from marine Chlorella species are more effective in activating immunity than fresh water Chlorella species. The polysaccharide extract was obtained via gel filtration with Sephadex G-50.
U.S. Pat. No. 4,786,496 discloses a lipid and glycolipid fraction of marine Chlorella with immuno-potentiating activity.
Kojima et al. J. Retic. Soc., 1973, 14:192-208 discloses a 1,250-1400 Da reticuloendothelial system (RES)-active glucan from Chlorella.
U.S. Pat. No. 3,462,412 discloses a process for preparing a RES-stimulating extract from Chlorella.
Japanese patent application, publication no. 06248003 discloses a Chlorella extract of 15 to 25 kDa, comprising polysaccharides containing predominantly galactose, with anti-neoplastic activity.
Mizuno et al. Bull. Fac. Agr. Shizuoka Univ. (Shizuoka Daigaku Nogakubu Kenkyu Hokoku), 1980, 30:51-59 discloses two fractions of neutral glycans from Chlorella, both apparently of small molecular weight.
Ukai et al. Ann. Proc. Gifu Pharm. Univ. (Gifu Yakka Daigaku Kiyo), 1990, 39:44-48 discloses two polysaccharides, CP-I and CP-II, from C. pyrenoidosa with RES-stimulating activity. CP-I comprises glucose, fucose, rhammose, galactose and mannose; CP-II comprises glucose, galactose, rhamnose and mannose.
Chu et al. Aquaculture, 1982, 29(3-4):241-252 discloses that the polysaccharide, ethanol-precipitable fraction of five algal species including Chlorella contains principally glucose, mannose, ribose/xylose, rhamnose and fucose.
Chlorella extracts prepared according to the invention show immune stimulatory activity in pharmacological and clinical tests. In one aspect, the extracts provided by the present invention have a higher immune stimulatory activity than extracts prepared and used in the art.
In one aspect, the invention provides preparations comprising high molecular weight Chlorella polysaccharide and polysaccharide complexes. The high molecular weight polysaccharide and polysaccharide complexes are about 1xc3x97105 Da to about 1xc3x97107 Da and constitute at least 22% (w/w) of the total Chlorella-derived content of the extract. In a preferred embodiment, the extract is derived from Chlorella pyrenoidosa. 
The high molecular weight polysaccharide and polysaccharide complexes may be of a selected range, e.g. about 1xc3x97105 Da to about 3xc3x97105 Da, about 3xc3x97105 Da to about 5xc3x97105 Da, about 5xc3x97105 Da to about 6xc3x97105 Da, about 6xc3x97105 Da to about 7xc3x97105 Da, about 7xc3x97105 Da to about 8xc3x97105 Da, about 8xc3x97105 Da to about 9xc3x97105 Da, about 9xc3x97105 Da to about 1xc3x97106 Da, about 1xc3x97106 Da to about 2xc3x97106 Da, about 2xc3x97106 Da to about 3xc3x97106 Da, about 3xc3x97106 Da to about 4xc3x97106 Da, about 4xc3x97106 Da to about 5xc3x97106 Da, about 5xc3x97106 Da to about 7xc3x97106 Da, about 7xc3x97106 Da to about 9xc3x97106 Da, and about 9xc3x97106 Da to about 1xc3x97107 Da.
The extracts may contain various different percentages of polysaccharide and polysaccharide complexes as a fraction of the total Chlorella-derived content of the extract. The percentage may be at least 24% (w/w), at least 26% (w/w), at least 28% (w/w), at least 30% (w/w), at least 35% (w/w), at least 40% (w/w), at least 45% (w/w), at least 50% (w/w), or at least 60% (w/w).
In another aspect, the high molecular weight polysaccharide and polysaccharide complexes contain glucose and any combination of: galactose, rhamnose, mannose and arabinose.
In another aspect, the high molecular weight polysaccharide and polysaccharide complexes is substantially free of ribose, nucleic acids, ribonucleic acids and unassociated proteins. The high molecular weight polysaccharide and polysaccharide complexes may also contain N-acetyl glucosamine and N-acetyl galactosamine.
In another aspect, the extracts of the invention retain immunomodulating activity upon treatment to remove unassociated DNA, RNA and proteins. Such treatment includes digestion by pronase, DNAse, RNAse and proteases.
In another aspect, the extracts of the invention retain immunomodulating activity upon treatment to effect cleavage of specific glycosidic linkages, the linkages being defined by their susceptibility to cleavage by amylase, amyloglucosidase, cellulase or neuraminidase. Such susceptible linkages are typically:
(i) three or more xcex1-1,4-linked D-glucose units;
(ii) xcex1-1,4-linked glucosides;
(iii) xcex1-1,4-linked galactosides; or
(iv) xcex1-1,4-linked D-glucose.
The invention also provides nutritional compositions containing the high molecular weight Chlorella extract with at least one energy source which may be carbohydrates, fats or nitrogen.
The extracts of the invention may also be used in combination or in mixture with a conventional supplement such as vitamin E, vitamin C and folic acid. The extracts may also be used with other nutraceuticals such as fish oils, spirulina and echinacea, especially those nutraceuticals which have immunostimulant activity.
The invention also provides a process for obtaining Chlorella preparations having immunomodulating activity. The process contains the steps of:
(a) size fractionating an aqueous extract of Chlorella, and
(b) selecting fractions comprising high molecular weight polysaccharide and polysaccharide complexes of about 1xc3x97105 Da to about 1xc3x97107 Da.
The process for obtaining the Chlorella extract may further include the step of pooling and concentrating the selected fractions. Size fractionation may be achieved by chromatography, ultrafiltration or ultracentrifugation.
The invention also provides a method for modulating the immune response of a mammal including humans by administering to the mammal an effective amount of the high molecular weight Chlorella extract. Such modulation includes increased proliferation of splenocytes and increased production of cytokines such as IL-6, IL-10, INF-xcex3 and TNF-xcex1, and may be advantageously used to treat or prevent bacterial or fungal infections.
The extract may further be administered as a supplement to a vaccination regimen to further stimulate the immune response. A flu vaccine may be advantageously used with the extract. The extract may be present as an adjuvant to the vaccines, especially as an oral vaccine adjuvant.